Enumeration of Bacteria Present in Food Samples

Introduction

In the many stages of bacteriology, the numerical detection of bacterial growth is important. In this experiment, the types of methods used to count bacteria are Microscopic Counts and Standard Plate Count (SPC). To aid in the counting of microbial growth, Petroff-Hauser chamber, a special slide with grid patterns, can be used in Microscopic Counts. For SPC, dead cells are excluded and only the amount of viable cells is measured (Tortora, 2003). Serial dilution is required to dilute the bacteria suspension before preparing it on an agar plate as counts over 300 would be difficult to measure (Talaro, 1993). Another method to count bacterial growth is to measure the turbidity. This method of enumeration is usually faster and is suitable for detecting microorganisms of high concentrations. The turbidity is measured using a spectrophotometer as the bacteria culture acts like colloidal suspension. Bacterial cells will absorb the light the passes through the culture at a defined wavelength and the number of present cells will decrease the light emerging from the culture proportionately. Both methods have advantages and disadvantages.

Microscopic Count has an advantage of being a faster method, as no incubation period is needed. It also does not require culturing and hence, selecting a preferred organism is not necessary. The morphology of the microorganism can also be observed when counting. Also, the count of microorganisms in dense suspensions can be carried out with proper dilution. The disadvantages of microscopic count include, both living and dead cells cannot be segregated; some cells may be missed as small cells are challenging to be seen under a light microscope; low density suspension is not suitable for this method, but centrifugation and filtration can concentrate the suspension to increase its sensitivity. However, it is very time-consuming, letting it become another limitation (Jennison, 1937).

For SPC, its sensitivity is regarded as an advantage as suspensions of low density can be used. Because of its sensitivity, inspection and correct identification of the counted microorganism can be carried out. It is also a less challenging method and the count of populations of any magnitude can be adapted. However, SPC only counts living cells and exclude dead cells. Clumping or chains of cells are developed which will hinder the accuracy of count of the number of cells present in relation to the total. SPC method is also more time consuming when compared to microscopic count, as incubation time is need for microorganisms to grow. The development of colonies depends on the microorganisms of which the condition of the culture is suitable for its growth (Jennison, 1937).

Microbial number is food samples are important for the safe consumption of food. Microorganisms produce bacteria, virus and toxins in which could contaminate food. Small amounts of these microorganism may cause food spoilage in which is unsafe for human consumption and can be a major concern to consumers and also suppliers. These microorganisms also serve as an indicator to identify the pathogenic species that is potentially present. Certain foods require high bacteria counts, but are not spoiled in order to ferment and produce probiotics. These probiotics are found to have health properties and are used to promote a healthy digestive system (Kechagia et al, 2013). These foods include yogurt and kimchi. In yogurt, the lactose fermentation by bacteria produces lactic acid in which caused yogurt to thicken from liquid milk and develop a sour flavour (Lourens-Hattingh & Viljoen, 2001). The main bacteria present in yogurt are Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus bacteria (Lee et al, 2012). Kimchi is a traditional Korean dish made by fermenting vegetables. The microorganism that dominated this food is Leuconostoc, Lactobacillus, and Weissella (Jung et al, 2011). Companies of beer and wine production monitor yeast growth during their process of distillation.

Objective

The objective of this experiment is to understand the methods to carry out bacteria count. To determine the increase in culture absorbance, a spectrophotometer is used and the values obtained are correlated with the values obtained from standard plate count. In this experiment, a standard plate count is performed to determine the number of bacteria present in food samples of kueh, sandwich, and curry puff. This experiment is also carried out to understand the importance of monitoring the bacteriology quality of foods.

Methods and Material

Part A: The Standard Plate Count

A bottle of nutrient agar is liquefied. While is being liquefied, three 9.9 sterile water blanks were labeled a, b, and c. Four petri plates were also labeled as 1:10,000, 1: 100,000, 1:1,000,000 and 1:10,000,000. As an addition, the amount to be pipetted into each plate, in which were 0.1ml or 1.0ml was labeled accordingly. The culture of E.coli was shaken and 0.1ml of the organism was transferred to blank a. Blank a was then shakes for 25 times to break up clumps of bacteria and also enforce good distribution. 0.1ml of blank a was then transferred into blank b using a different pipette. In the same manner as was done with blank a, blank b was shaken for 25 times. Another sterile pipette was use used to transfer 0.1ml of blank b into the 1:100,000 plate and 1.0ml was transferred into 1:10,000 plate. Then, with a different pipette, 0.1ml from blank b was transferred to blank c. Blank c was also shaken for 25 times. Using another sterile pipette, 0.1ml of blank c was transferred into the 1:10,000,000 plate and 1.0ml into the 1:1,000,000 plate. Once the bottle of nutrient agar has boiled for 8 minutes, it is cooled in a water bath at 50C for 10 minutes. One-forth of the nutrient agar, in which is 20ml, is poured into the four plates each. The plates were gently rotated to achieve adequate mixing of the medium and organisms. After the medium was cooled completely, it was then incubated at 35C for 48 hours in an inverted position.

Part B: Determination of Growth by Absorbance (optical density)

Four test tubes are labeled 1:2, 1:4, 1:8 and 1:16. Using a 1ml micropipette, 1ml of sterile nutrient broth was dispense into each tube with labels 1:2, 1:4, 1:8 and 1:16. The culture of E. coli was shaken vigorously to suspend the organism. Using the same micropipette, 1 ml of E. coli was transferred into the 1:2 test tube. The contents in the 1:2 tube was mixed by drawing the mixture up into the pipette and discharging it into the tube three times. Then, 1ml from the 1:2 tube was transferred to the 1:4